AFJOG

African Journal of Obstetrics and Gynaecology | Volume 2 | Issue 2 | 2024 | 34 CASE REPORT African Journal of Obstetrics and Gynaecology | Volume 2 | Issue 2 | 2024 | Polycystic Ovary Syndrome: An update from the 2023 international guideline New onset adrenal failure may cause undetectable 17- OHP in an individual with previously elevated levels. In one case report a boy who was diagnosed at birth with CAH secondary to 21OHD based on elevated 17-OHP and genetic analysis, subsequently developed autoimmune adrenalitis resulting in undetectable 17-OHP from the second year of life due to adrenal failure (6) . Other potential causes of subsequent adrenal failure are infarction following adrenal haemorrhage, adrenoleukodystrophy, infection with cytomegalovirus, and prolonged high dose glucocorticoid therapy (6) . Our patient was not on medical therapy and had no evidence of complete adrenal insufficiency both clinically and biochemically. We could not find proof that an inadvertent sample swap or mislabelling may have contributed to the false negative result, though we cannot completely rule this out. The blood was collected in a designated specimen collection room at the hospital. Patients are typically consulted individually in separate rooms. The time from specimen collection to commencement of laboratory processing was less than 30minutes. Two yellow top tubes labelled with her name arrived together in the laboratory. One was processed for 17-OHP and frozen within 60minutes of arrival in the laboratory. The other was used to test for DHEAS and electrolytes, results of which were consistent with our patient’s clinical picture and her subsequent test results. There were no reports from other clinicians of inconsistent results for other patients who were tested for 17-OHP in the same batch. There was also no other patient from the same processed batch who had extremely elevated results such as the ones subsequently observed from our patient. A competitive radioimmunoassay (Beckman RIA 17α-hydroxyprogesterone (REF 1452)] was used for analysis instead of LC-MS/MS. Failure to accurately follow the steps in the instruction manual may bring about false negative results. Our laboratory has a standing policy not to analyse hemolysed, lipemic or icteric samples for 17-OHP as per recommendations. The sample was frozen and batched for analysis. Freezing and thawing of samples, up to ten repeated cycles, does not seem to significantly affect 17- OHP results (7). The manual allows for storage of specimen up to one year below -18°C after aliquoting. High dose hook effect (HDHE) while using an enzyme- linked immunosorbent assay for 17-OHP testing was documented in one case report (8) . HDHE generally occurs in sandwich immunoassays in which both capture and marker antibody are added simultaneously without a wash step resulting in saturation of both sets of antibodies by analyte and failure of sandwich formation. The phenomenon may still occur even with a wash step in cases of very high analyte concentration (9,10) . Sample dilution usually resolves this problem. HDHE is typically not a concern in competitive immunoassays (11) such as the one used for our patient. RIA depends on the mass effect of the analyte in the patient sample displacing the radiolabelled 17-OHP from the capture antibody. After washing, the radioactivity of bound analyte and supernatant are measured and interpolated onto a standard curve (12) . HDHE remains a possibility as it is well described in settings such as molar pregnancy where there are excessive levels of circulating hormone (9) . The presence of autoantibody against an analyte or reagent may positively or negatively affect immunoassays (9,11) . Co-existent natural biological components within a sample, or components within the test tubes and testing kits may have low level binding to analytes of interest or antibody resulting in interference via the matrix effect, causing inter and intra-individual variability of results (11) . Falsely elevated 17-OHP may be a result of non-specific binding of antibody to other similar steroids such as 17α-hydroxypregnenolone or medications with steroid structure such as spironolactone. Cross-reactivity may be attenuated by extraction of 17α-hydroxypregnenolone with ether prior to 17-OHP testing. Extraction with ether was one of the steps of the assay used in our laboratory and the patient was not on any medications at the time of presentation. LC-MS/MS is more specific. Immunoassays continue to be used for screening, and point of care testing because they are cheaper, quicker, simpler and less labour intensive to perform (3,13) . True elevations during stress or sickness and prematurity in infants may also result in elevated 17-OHP (3) . Heterophile antibodies or human anti-animal antibodies in individuals who have received prior immunotherapy or who regularly handle animals may cause false positive results. Diluting the samples or blocking the heterophile antibodies usually eliminates this interference (11) . Pipetting errors are possible. Failure of quality control may also occur in which case the test run should fail and no results released. Post-analytical errors could occur during transcription and verification of results (5) . All results processed in the same batch as our patient were rechecked and there was no unusually elevated result for another patient which could suggest mismatching of results to patient. Because 21OHD is the commonest cause of virilising CAH, we repeated the test. Clinicians should collaborate with the laboratory by providing adequate clinical information when they request investigations, interpret results in conjunction with other clinical features and liaise with the laboratory in case of spurious results (9) . Other forms of virilising CAH may cause elevation of 17-OHP viz, 11βHD, 3βHSD2 deficiency and p450 oxidoreductase deficiencies. The clinical presentation and an ACTH stimulation test may assist to differentiate the enzyme deficiencies. Our patient had 21OHD based on marked elevation of 17-OHP. Increases in 11 deoxycortisol may occur in patients with partial 21OHD, related to overwhelming production of upstream precursors. In our resource- limited setting, pursuing further expensive biochemical and genetic testing to confirm the enzyme defects was not a consideration because it would not have altered further management of the patient. Genotyping should be reserved for equivocal situations and when accurate ACTH stimulation testing cannot be performed; or to facilitate genetic counselling (3) . Our patient and family did not fully understand the investigations, diagnosis and treatment she had received in childhood nor did they have any treatment records. Individuals with disorders of sex differentiation should be managed by a multidisciplinary team. The optimal plan would include offering the family extensive counselling regarding the diagnosis, results of investigations, and long term treatment plan as the affected individual transitions to adulthood (3) . Patients with adrenal insufficiency should carry medical identification which may be in the form of written documentation or medical alert bracelets and hydrocortisone for stress dosing in emergency situations (3) .